PROJECT SUMMARY Understanding the autoimmune response in human type 1 diabetes (T1D) is crucial for the design of successful immunotherapies for those at-risk for the disease as well as for those with established disease. While studies of rodent models of have greatly instructed us on the autoimmune process, the translation of successful therapies and prevention studies in the rodent models to human clinical trials has been far less successful. While we know much information concerning islet infiltrating T cells in the rodent models and islet infiltrates in humans with T1D by histology and immunohistochemistry, we previously have had limited opportunity to isolate directly the lymphocytic infiltrate from humans with T1D for studies of repertoire analysis and functional capacity. We have received islet isolations from 12 individuals with T1D and have grown or sorted directly out of the islets, ~300 CD4+ and CD8+ T cell lines or clones. We have demonstrated the reactivity and HLA restriction of 16 CD4+ T cell lines or clones and 3 CD8+ T cells lines (multiple reactivities are represented). The purpose of the study proposed here is to comprehensively define the islet reactivity, HLA restriction and extensive phenotype, RNA transcriptome, T cell receptor structure and T cell receptor repertoire analysis of the existing, but yet, uncharacterized islet infiltrating T cells, with an emphasis on islet-infiltrating CD8+ T cells. These studies will include T cells derived from the islets of donors with T1D which we anticipate receiving during this funding period. Islet-infiltrating T cells will be used as tools for the immunopathologic study of T1D and importantly, for comprehensive understanding of the function of islet-infiltrating T cells in T1D immunopathology. Our hypothesis is that-islet infiltrating CD4+ and CD8+ T cells will recognize a broad range of peptide ligands, resulting from both islet-specific expression and islet-specific post-translational modification/splicing, using restricted T cell repertoires, but expressing a similar proinflammatory, memory phenotypes. These islet- infiltrating T cells will be vital tools/reagents for investigators in the field to study the function of islet-infiltrating T cells in humanized mouse models of T1D, T cell receptor repertoire analysis, for preservation of autoreactive T cell receptors for future studies, in functional assays of autoreactive T cell interactions with T regulatory cells, B cell, antigen presenting cells and the innate immune system. Finally, these studies will inform investigators in the design of successful immunotherapies for those at-risk for T1D and especially for those with established T1D. The studies will continue our successful collaborations as part of HIRN and with HIRN investigators.